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| United States Patent |
5,698,398
|
|
Shassere
, et al.
|
December 16, 1997
|
Control compositions for determination of molecular cytogenetic
abnormalities with DNA probes
Abstract
Quality control compositions suitable as sample specimens to measure
performance of DNA probe tests which determine cytogenetic abnormalities,
such as chromosome copy number, of cells in a tissue sample are disclosed.
The control compositions comprise a suspension of fixed cells having an
artificial concentration of cells exhibiting chromosome specific
aneuploidy in one or more specific chromosomes.
| Inventors:
|
Shassere; Christine J. (1613 Preston Rd., Naperville, IL 60565);
Seelig; Steven A. (2409 Remington Dr., Naperville, IL 60565)
|
| Appl. No.:
|
538768 |
| Filed:
|
October 5, 1995 |
| Current U.S. Class: |
435/6 |
| Intern'l Class: |
C12Q 001/68 |
| Field of Search: |
435/6,240.2,240.3,810,240.25
|
References Cited
U.S. Patent Documents
| 5059518 | Oct., 1991 | Kortright et al. | 435/6.
|
| 5409826 | Apr., 1995 | Maples et al. | 435/240.
|
Other References
van Dekken et al., Cancer 66, 491-497 (1990).
Klinger et al., Am. J. Hum. Genet. 51, 55-65 (1992-Jul.).
Paxton et al., Int. Soc. Analy. Cytol., 16 (Mar. 1993).
|
Primary Examiner: Horlick; Kenneth R.
Parent Case Text
This is a continuation of application Ser. No. 08/139,351, filed Oct. 18,
1993, abandoned.
Claims
We claim:
1. A control composition to measure performance of a test for determining
molecular cytogenetic abnormality in individual cells comprising:
(i) a suspension media, and
(ii) a fixed cell mixture comprising
(a) cells which are from at least two cell sources and which have similar
morphology in their cell size, cell shape and cell configuration, wherein
(1) at least one cell source exhibits at least one molecular cytogenetic
abnormality in at least one chromosome and (2) likelihood of visual
distinction between the cell sources by an observer is reduced,
(b) a minimum total cell concentration above about 10,000 cells per
milliliter, and
(c) an artificial concentration of abnormality-containing cells exhibiting
at least one molecular cytogenetic abnormality in at least one chromosome.
2. The control composition of claim 1 wherein the fixed cell mixture
comprises cells from each of two human tumor cell sources.
3. The control composition of claim 1 wherein the artificial concentration
of abnormality-containing cells is about 5 to about 95 percent of total
cells in the cell mixture.
4. The control composition of claim 1 wherein the abnormality-containing
cells exhibit chromosome specific aneuploidy in at least one chromosome.
5. The control composition of claim 4 having an artificial concentration of
abnormality-containing cells which exhibit trisomy 8.
6. The control composition of claim 1 wherein the molecular cytogenetic
abnormality comprises at least one of a chromosomal sequence
translocation, a chromosomal sequence deletion or a chromosomal sequence
amplification.
7. The control composition of claim 4 wherein the aneuploid cells exhibit
chromosome specific aneuploidy in a chromosome selected from the group
consisting of chromosomes 1 through 22, X and Y.
8. A control composition to measure performance of a test for determining
chromosomal copy number of individual cells comprising:
(i) a suspension media, and
(ii) a fixed cell mixture comprising
(a) cells which are from at least two cell sources and which have similar
morphology in their cell size, cell shape and cell configuration, wherein
(1) at least one cell source exhibits chromosome specific aneuploidy in at
least one chromosome and (2) likelihood of visual distinction between the
cell sources by an observer is reduced,
(b) a minimum total cell concentration above about 10,000 cells per
milliliter, and
(c) an artificial concentration of aneuploid cells exhibiting chromosome
specific aneuploidy in at least one chromosome.
9. The control composition of claim 1 wherein the fixed cell mixture
comprises cells from each of at least two human tumor cell sources.
10. The control composition of claim 1 having an artificial concentration
of aneuploid cells which exhibit trisomy 8.
11. The control composition of claim 1 wherein the artificial concentration
of aneuploid cells is about 5 to about 95 percent of total cells in the
cell mixture.
12. The control composition of claim 1 wherein the aneuploid cells exhibit
chromosome specific aneuploidy in two or more chromosomes.
13. The control composition of claim 8 wherein the aneuploid cells are
derived from a human tumor cell line.
14. The control composition of claim 8 wherein each of the cell sources
comprises human tumor cells.
15. The control composition of claim 14 wherein the aneuploid cells exhibit
chromosome specific aneuploidy in a chromosome selected from the group
consisting of chromosomes 1 through 22, X and Y.
16. A container containing a control composition useful to measure
performance of a test for determining chromosomal copy number of
individual cells in a tissue sample comprising:
(i) a suspension media, and
(ii) a fixed cell mixture comprising
(a) cells which are from at least two sources and have similar morphology
in their cell size, cell shape and cell configuration, wherein (1) at
least one cell source exhibits at least one molecular cytogenetic
abnormality in at least one chromosome and (2) likelihood of visual
distinction between the cell sources by an observer is reduced,
(b) a minimum total cell concentration above about 10,000 cells per
milliliter, and
(c) an artificial concentration of abnormality-containing cells exhibiting
a molecular cytogenetic abnormality in at least one chromosome.
17. The container of claim 16 wherein the abnormality-containing cells are
human cells exhibiting chromosome specific aneuploidy in a chromosome
selected from the group consisting of chromosomes 1 through 22, X and Y.
18. The container of claim 16 in a kit comprising at least one DNA probe.
19. A method of manufacturing a control composition to measure performance
of a test for determination of molecular cytogenetic abnormalities,
comprising: a cell suspension having an artificial concentration of
abnormality-containing cells exhibiting a specific molecular cytogenetic
abnormality, which method comprises:
(a) mixing a first cell source with at least a second cell source in an
appropriate ratio to produce an intermediate cell suspension, wherein (i)
each of the first and second cell sources comprise cells in a suspension
media which does not inhibit cell growth and cell concentration of the
first and second cell sources is approximately equal, (ii) one of the cell
sources comprises a predetermined artificial concentration of cells
exhibiting the specific molecular cytogenetic abnormality and (iii) the
first and second cell sources comprise cells having similar morpholgy in
their cell size, cell shape and cell configuration;
(b) treating the intermediate cell suspension by addition of a cell
fixative solution to produce a control composition comprising fixed cells
in suspension in a suspension media which does not sustain cell
multiplication; and
(c) determining the percent of total cells in the control composition
exhibiting the artificial concentration of cells exhibiting the specific
abnormality for a selected chromosome.
20. The method of claim 19 wherein the specific molecular cytogenetic
abnormality is chromosome specific aneuploidy in at least one chromosome.
21. The method of claim 19 which further comprises: (d) producing a
hybridization slide from the control compositions.
22. The control composition produced by the method of claim 19 comprising a
minimum total cell concentration above about 10,000 cells per milliliter,
wherein the abnormality-containing cells comprise cells from a human tumor
cell line.
23. The control composition of claim 22 wherein the cells in the control
composition exhibit chromosome specific aneuploidy in a chromosome
selected from the group consisting of chromosomes 1 through 22, X and Y.
24. The control composition of claim 22 having an artificial concentration
of cells exhibiting chromosome specific aneuploidy in the range of 5 to 95
percent of total cells in the control composition.
25. A microscope examination slide comprising the control composition of
claim 1.
26. A microscope examination slide comprising the control composition of
claim 8.
27. A microscope examination slide comprising the control composition of
claim 22.
Description
FIELD OF THE INVENTION
This invention relates generally to quality control standards for use with
in-situ hybridization procedures using DNA probes, and more particularly
to control compositions comprising an artificial concentration of cells
exhibiting at least one molecular cytogenetic abnormality, which are
useful as quality control standards for DNA probes.
BACKGROUND
Molecular cytogenetics is the evaluation and determination on a molecular
level of an organism's genetic material, such as the DNA contained in
human chromosomes. Molecular cytogenetics is of growing research and
clinical significance because of the recognition that cancer and other
diseases result from changes in the DNA of a chromosome or from the
inherited DNA make-up of a chromosome. Molecular cytogenetics involves
attempts to establish correlations between specific diseases and genetic
events, such as the existence of an abnormal number of a particular
chromosome, a condition called aneuploidy. For example, acute myelogenous
leukemia has been correlated to the presence in the affected blood cells
of three copies, compared to the normal two copies, of chromosome 8, a
condition called "trisomy 8". Therefore, molecular cytogenetic analysis,
such as methods for chromosome enumeration, is of diagnostic and
prognostic significance to the physician.
Fluorescence In Situ Hybridization ("FISH") is a molecular cytogenetic
technique of increasing use. FISH involves the use of DNA probes to
determine if a particular nucleotide sequence is present in the
chromosomal DNA of particular cells. These DNA probes comprise a
nucleotide sequence complementary to a specific target DNA nucleotide
sequence in a chromosome, wherein the probe is directly labeled with a
fluorescent molecule or indirectly labelled with an antibody which can be
bound to a fluorescing material. In use, the DNA probe is mixed with the
cell sample under conditions which permit hybridization of the nucleotide
sequence of the DNA probe to its corresponding target nucleotide sequence
in the chromosome, if present, to form a hybrid reaction complex. If
formed, the hybrid reaction complex is subsequently detected with a
fluorescent microscope. An example of commercially available DNA probes
for FISH use is the SpectrumCEP.TM. Chromosome Enumeration Probes
available from Imagenetics (Framingham, Mass.). These chromosome
enumeration probes each comprise direct fluorescently labelled DNA probes
containing satellite DNA sequences (alpha, beta satellite III) which are
commonly complementary to the centromere region of a particular
chromosome. In addition to being complementary to the centromere, these
satellite DNA sequences may be complementary to other parts of the
particular chromosome. These chromosome enumeration probes thus permit
accurate determination of chromosome specific aneuploidy (chromosomal copy
number) in the cells of a particular tissue sample.
One problem for the use of FISH to determine chromosomal copy number is the
absence of any commercially available control standards to verify correct
analysis. The U.S. Clinical Laboratories Improvement Act of 1990 also
mandates that clinical testing laboratories perform certain quality
control analysis in relation to tests performed by the clinical
laboratories. With regard to control standards for chromosomal copy number
determination, the only presently available control technique is to have
on hand appropriate cell lines of known chromosomal aneuploidy which can
be used to calibrate the technique and analysis used. This is not
practical for the majority of clinical laboratories which will not have
access to appropriate cell lines. There is therefore a need for control
test standards having "artificial" concentrations of aneuploid cells which
can be used to calibrate chromosome enumeration analysis.
Applicants are not aware of any literature reference describing the use of
test standards having artificial concentrations of aneuploidy cells for
use in calibration of chromosome enumeration techniques. A cell mixture
for use in FISH research is disclosed in "Detection of Male Cells in
Mixtures Containing Varying Proportions of Male and Female Cells by
Fluorescence In Situ Hybridization and G-Banding", A. White et al.,
CYTOMETRY, Vol. 14, pp. 9-15, 1993. The disclosed cell mixtures contained
normal cells and were used to assess the ability of FISH to detect male
cells. These mixtures did not comprise cells containing a molecular
cytogenetic abnormality.
"Use of Aneuploid Cell Lines as Standards and Calibrators in DNA Analysis",
H. Paxton, et al., International Society for Analytic Cytology: 1993
Abstracts, page 16, published March 1993, describes "standard
preparations" which contain tumor cells mixed with human peripheral blood
lymphocytes ("PBL's"). These preparations are not disclosed for use with
molecular cytogenetic determination of a chromosome specific abnormality,
such as chromosome specific aneuploidy. The described standard
preparations were used in evaluation by flow cytometry for the percentage
of aneuploid recovery, aneuploid peaks and reproducibility of assay value.
Flow cytometry is only capable of gross chromosomal aneuploidy
measurement; it does not determine whether specific chromosomes are
aneuploid. Furthermore, the standard preparations disclosed therein are
cells with grossly different morphologies. The different morphologies make
the disclosed preparations unsuitable in chromosomal copy number
determination because of possible observer bias in the results.
Chromosomal copy number determination involves visual examination; cells
with grossly different morphology can lead to inaccurate analysis.
Finally, the Abstract does not disclose the method of manufacture nor does
it disclose control compositions comprising tumor cells from two or more
sources.
Therefore, control compositions useful in conjunction with determinations
of specific molecular cytogenetic abnormalities on a specific chromosome,
such as chromosomal copy number determinations with DNA probes, are not
available nor disclosed. It is an object of the invention to provide
control compositions having an "artificial" concentration of cells with
chromosome specific abnormalities suitable to measure performance of
molecular cytogenetic techniques. It is a further object to provide
methods for manufacture of such control compositions. Another object is to
provide control compostions for performance measurement of chromosome
enumeration DNA probes. Other objects will appear below.
SUMMARY OF THE INVENTION
The invention broadly comprises control compositions suitable to measure
performance of tests which determine specific molecular cytogenetic
abnormalities on specific chromosomes of individual cells, comprising a
suspension media and a fixed cell mixture which comprises: (i) cells from
at least two sources having similar morphology, (ii) a total cell
concentration above about 10,000 cells per milliliter of control
composition and (iii) a predetermined, "artificial" concentration of
abnormality-containing cells exhibiting at least one molecular cytogenetic
abnormality in at least one chromosome. The inventive compositions are
used as sample specimens and provide clinical and research laboratories
using techniques to measure cytogenetic abnormalities, such as FISH
performed with chromosome enumeration DNA probes, with the ability to
verify proper performance of their analytic techniques. The control
compositions can comprise cells with chromosome specific aneuploidy for
each human chromosome and therefore serve as controls for a wide number of
chromosome enumeration tests. Preferably, the control compositions
comprise fixed human cells having an artificial concentration of cells
exhibiting aneuploidy in at least one of chromosomes 1 through 22, X and
Y. The inventive compositions can further comprise two or more different
types of abnormality-containing cells, each type exhibiting a different
chromosome specific abnormality, thereby being suitable as a simultaneous
control for more than one DNA probe.
The invention further comprises a method for the manufacture of the
inventive compositions. Applicants' early attempts to manufacture the
inventive compositions involved determination of the cell count for each
of two different cell sources, one having a high percentage exhibiting
aneuploidy in a desired chromosome cell (the "abnormal" source) and the
other having a low percentage of cells exhibiting aneuploidy in a desired
chromosome (the "normal" source), each source comprising cells in
suspension in a fixative solution. However, addition of the fixative
solution to each source prior to mixing of the two cell sources resulted
in suspensions for which Applicants were unable to determine cell
concentration. Applicants found, instead, that the inventive compositions
could be made by a method comprising: mixing a first cell solution with a
second cell solution, each of said cell solutions having a known cell
concentration and comprising a media capable of sustaining cell
multiplication, in a desired ratio to produce an intermediate mixed cell
suspension; treating the intermediate suspension to produce a control
composition comprising fixed cells; and determining the chromosome
specific abnormality content of cells in the control composition.
The method of the invention can be used to produce a predetermined,
"artificial" concentration of cells exhibiting specific molecular
cytogenetic abnormality content in one or more chromosomes, by selection
of the appropriate first and second cell sources and by mixing of the cell
sources in the desired ratios. The inventive method has the further
advantage of reliable reproduction of the inventive compositions.
DETAILED DESCRIPTION OF THE INVENTION
The compositions of the invention comprise an artificial concentration of
cells exhibiting at least one molecular cytogenetic abnormality in at
least one chromosome. The control compositions comprise an "artificial"
concentration of cells exhibiting the cytogenetic abnormality, which is
preferably chromosome specific aneuploidy. By artificial concentration,
Applicants mean that the control composition has a preselected
concentration of cells, based on total cell content of the composition,
which exhibits the specific cytogenetic abnormality in the specified
chromosome. The control compositions can also comprise cells exhibiting:
molecular cytogenetic abnormalities in more than one chromosome, more than
one molecular cytogenetic abnormality in the same chromosome, or any
combination thereof. The types of cytogenetic abnormalities include DNA
sequence translocations between chromosomes, DNA sequence deletions from a
chromosome, DNA sequence amplification on a chromosome, and chromosomal
aneuploidy. As is known in the art of molecular cytogenetics, even normal
cell populations contain low ("background") percentages of cells which
contain cytogenetic abnormalities. The background level varies with the
specific abnormality, but is believed generally less than 2.0% of total
cells in the population. Thus, in the control compositions of the
invention, the artificial concentration of cells with an abnormality is
any preselected percentage, based on total cells in the control
composition, which is greater than the background level of normal cells
exhibiting the abnormality. The control compositions comprise cells from
at least two cell sources, one of low ("normal") content of cells
exhibiting the specific cytogenetic abnormality, the other of higher
("abnormal") content of cells exhibiting the specific abnormality.
The inventive compositions preferably comprise control standards useful to
measure the performance of chromosome enumeration DNA probes, such as the
SpectrumCEP.TM. probes from Imagenetics (Framingham, Mass.). The control
compositions of the invention comprise cells derived from a minimum of two
sources, one cell source having a "normal" aneuploidy content and the
second having an "abnormal" aneuploidy content. Human cells are normally
diploid, i.e. they each contain two copies of chromosomes 1-22 and one
pair of sex chromosomes, X and Y. Any number of chromosomes other than two
of each chromosome per cell is termed aneuploid. However, even healthy,
normal human cells contain a low background percentage of cells exhibiting
aneuploidy in one or more chromosomes. As used herein, normal aneuploidy
content means that the cells exhibit only a low background concentration
of cells exhibiting chromosome specific aneuploidy in one or more
chromosomes. The background level is preferably less than about 2.0% of
total cells exhibiting aneuploidy in the desired chromosome. The second
cell source contains a significant percentage of cells exhibiting
chromosome specific aneuploidy in the desired chromosome or chromosomes.
Thus, the abnormal aneuploidy content is the desired percentage of cells
exhibiting the specific chromosome anueploidy. Further, the second cell
source can have individual cells exhibiting aneuploidy in more than one
chromosome. The use of two or more cell sources to produce the inventive
compositions permits adjustment of the abnormality-containing cell content
of the control composition to any desired level for any desired chromosome
based on cell source selection and on the mixing ratios of the two or more
cell sources.
Each of the "normal" and "abnormal" cell sources is from cell lines or cell
sources having similar cell morphology, i.e. size, shape and
configuration. The reason for this is that the control compositions are
primarily used in conjunction with FISH DNA probes which are analyzed with
the aid of a fluorescent microscope; the cells in the control should not
have widely different cell morphology, so as to avoid introducing observer
bias. The similarity of cell morphology for the cell sources is visually
assessed. When the compositions comprise more than two cell sources, the
likelihood of observer bias can be reduced by having at least two of the
cell sources having similar morphology.
The cell sources can be of any source which comprises cells sustainable in
vitro by in vitro culture techniques. The cell sources can comprise human
bone marrow cells, lymphoid or myeloid, ascitic cells from intraabdominal
fluid, cells from pleural effusions, fibroblasts, and cells from solid
tumors, preferably primary solid tumors. Preferably the cell sources
comprise bone marrow cells, ascitic cells or cells from pleural effusions,
because these cells are easier to culture.
Any suitable cell line or cell source can be used for either the first or
second cell sources. Preferably, the cell sources are human cell lines,
with the source from a tumor cell line having an abnormal aneuploidy
content. For example, a cell line with a high concentration of cells
exhibiting trisomy 8 can be used to produce a control composition having
an artificial concentration of trisomy 8 cells. Such a cell line is the
K-562 tumor cell line available as ATCC Deposit No. CCL-243 from the
American Tissue Culture Collection (Rockville, Md.). A cell source having
a normal background level of aneuploidy in chromosome 8 is the cell line
GM06912A, obtained from the NIGMS Human Genetic Mutant Cell Repository
(Camden, N.J.). More preferably, each of the first and second cell sources
comprise human tumor cells of differing chromosome specific aneuploidy, so
that the resulting control compositions are controls for more than one
type of chromosome specific aneuploidy tests. The inventive compositions
can also comprise cells from more than two sources, for example, a cell
source of normal aneuploidy content in each of two chromosomes, and two
other cell sources, each of abnormal anueploidy content in one of the two
chromosomes.
Applicants do not believe it necessary to perform a preliminary cell
sorting of the cell sources to ensure that substantially all cells in each
cell source are at substantially the same stage in their cell cycles.
However, it is within the scope of the invention to use cell sources which
comprise cells which are substantially within the same cell cycle stage to
produce the inventive compositions. The necessary cell sorting to produce
such cell sources can be performed by flow cytometry.
Any suitable artificial concentration of the abnormality-containing cell
content can be used and preferably will comprise 5 to 95 percent
abnormality-containing cells, based on total cells in the composition.
This range is preferred because a preferred control composition is one
comprising cells from one source with a 95% concentration of cells
exhibiting a first abnormality and a second source with cells exhibiting
95% concentration of cells with a second abnormality. Preferably the
artificial concentration is one of three concentrations: about 5% total
cell content, about 10% total cell content and about 50% of total cell
content. Thus the inventive compositions comprise artificial
concentrations of cells exhibiting a specific molecular cytogenetic
abnormality in a particular chromosome in the range of about 4 to about 6
percent of total cell content, about 8 to about 12 percent of total cell
content, and about 48 to about 52 percent of total cell content. Other
abnormal artificial cell concentrations are possible, if desired. Further,
the control compositions can comprise cells exhibiting an artificial
concentration of aneuploid cells for one chromosome, and a second
artificial concentration of cells exhibiting aneuploidy in a separate
chromosome or chromosomes.
The control compositions are prepared using sufficient suspension media to
produce a minimum cell concentration which, when the composition is used
to prepare a glass microscope hybridization slide, results in a cell count
of 500 cells per slide. Preferably the cell concentration in the control
composition is sufficient to produce a slide count of at least 1,000 cells
per slide. These mounts are believed to be of above about 10,000 cells per
milliliter of control composition (for 500 cells/hybridization slide) and
above about 20,000 cells per milliliter of control composition (for 1000
cells/hybridization slide). This minimum amount is preferred because of
the intended use of the compositions: cell concentrations below this
amount can lead to inaccurate hybridization analysis resulting from too
few cells on the hybridization slide.
The inventive compositions are used as control standards in the measurement
of the performance of techniques for determination of molecular
cytogenetic abnormalities like chromosomal copy number. The control
compositions are particularly suitable as control standards for use with
chromosome enumeration DNA probes for FISH which determine chromosome copy
number of cells in a particular specimen tissue sample. The control
compositions comprise cell suspensions which are handled similarly to
tissue samples undergoing analysis in a clinical or research laboratory.
FISH tests employing chromosome enumeration DNA probes involve the
preparation of hybridization slides containing the tissue sample to be
tested, to which is applied the chromosome enumeration DNA probe under
conditions which permit hybridization between the DNA of the tissue sample
with the DNA probe. After the hybridization reaction has occurred, the
slide is then washed to remove any unreacted probe, and is then analyzed
to determine the presence of the hybrid reaction complex comprising the
probe and its target nucleotide sequence. Typically the analysis for the
presence of the hybrid reaction complex is with the aid of a fluorescent
microscope. Similarly, the inventive compositions are used as specimen
samples to produce hybridization slides in which chromosomes in the cells
in the inventive composition serve as the target for the chromosome
enumeration DNA probe. The slides produced from the inventive composition
are then tested with the chromosome enumeration probe using any suitable
hybridization technique, which for quality control purposes is preferably
the identical technique to that used by a laboratory for testing unknown
tissue samples. Because the control compositions can be reproduced from a
wide variety of cell sources, they can be used as a quality control for
use of a wide variety of DNA probes. Use of the control compositions
permits the laboratory to assess variables in its use of DNA probes. For
example, assessment of temperature affect on hybridization can readily be
made.
The invention also comprises a method of manufacture of control
compositions for perfomance measurement of chromosome enumeration DNA
probes. The manufacturing of the inventive composition begins by obtaining
cells from appropriate sources for use as the first and as the second cell
source. Each of the cell sources is then counted by any suitable method,
such as with a hemocytomer or, preferably, a Coulter counter, to determine
cell count of the particular cell source. This data is needed to calculate
the appropriate ratio of amounts of the first cell source and amounts of
the second cell source to be combined. The percent cells exhibiting the
selected chromosomal abnormality, i.e. aneuploidy, in each of the first
and second cell sources is then determined. Preferably the first cell
source exhibits only a low background level of aneuploidy in the
chromosome of interest. The low background level is typically less than 2
percent. Once the cell counts and aneuploidy content of the cell sources
are known, they can be mixed in a desired ratio to produce the desired
control compositions.
As seen in Comparative Example 1, Applicants determined that mixing of
first and second cell sources which had been individually treated with a
fixative solution before their combination, was not feasible, because the
cell count of the individual cell sources could not be determined due to
excessive cell clustering. Therefore, the inventive compositions are
produced by addition of a fixative solution, to be used as the suspension
media for the inventive compositions, only after the first and second cell
sources have been mixed in the desired ratios.
Typically, sources of human cells are available in a media which permits
cell multiplication. Applicants prefer to obtain cells in a known growth
media comprising electrolytes such as NaCl, KCl, phosphates and a pH
buffer salt, for example, minimum essential media (MEM) available from
GIBCO/BRL, for use to produce the inventive compositions. The appropriate
volume of the cell source is mixed in MEM to produce the first and second
cell sources. At the time of determination of the cell count of the cell
sources, the growth media does not comprise serum or other additives which
aid cell multiplication.
After determining the cell counts and the molecular cytogenetic abnormality
content, the first and second cell sources are mixed in a desired ratio to
produce an intermediate cell suspension. The intermediate cell suspension
is then treated under suitable conditions, for example by centrifuging, to
remove the existing growth media in the intermediate cell suspension.
After removal of the growth media, the cell pellets are each treated with
a hypotonic solution to cause cell swelling. A suitable hyoptonic solution
is aqueous KCl solution. After treatment, the hypotonic solution is
removed by centrifuging and removal of the supernatent. The resulting
cells are then dissolved into a fixative media such as Carnoy's fixative
to produce the cell suspension of the invention.
Any suitable fixative solution can be used as the suspension media of the
inventive compositions. It is preferred to use a blend of 3/1 by volume
methanol and acetic acid because this fixative solution is readily
available as Carnoy's fixative and works well.
Any suitable mixing procedure can be used to produced a homogeneous mixture
of the cells in the fixative solution. The mixing solution should not be
so disruptive as to unduly disturb the cell nuclei. After the cell
suspension has been produced, the percent chromosome specific aneuploidy
content or other abnormality of this control composition is determined,
preferrably by use of a chromosome enumeration probe such as the
SpectrumCEP.TM. chromosome enumeration probe available from Imagenetics,
Inc.
In another embodiment the method of the invention further comprises
production of hybridization slides comprising the control compositions of
the invention. Any suitable slide preparation procedure can be used.
Preferrably the control composition is dropped onto a microscope
examination slide and stored under nitrogen at -20.degree. C. to await
use. The actual amount of control composition added to the hybridization
slide is any desired amount and is typically 100 microliters or about 1
drop from a a two milliliter Pasteur pipette.
In another embodiment, the invention comprises a container comprising the
control compositions. When packaged in appropriate containers, the control
compositions can be transported to customer clinical and research
laboratories for use and storage. Any suitable container or package for
storing of the cell suspension comprising the fixed cells and suspension
media of the invention can be used. Preferrably the containers in this
embodiment of the invention comprise a nonreactive, opaque, plastic
container of suitable size and having air tight seals. The containers
comprising the control compositions can be included in kits, for example,
containing more than one type of control composition or containing DNA
probes.
The following Examples are illustrative, but not limiting, of the
invention.
EXAMPLE 1
A sample of the continuous cell line K-562 was obtained from the American
Tissue Culture Collection, Deposit ATCC CCL-243. The K-562 cell line was
established from the cells of a fifty-three year old human female with
chronic myelogenous leukemia in terminal blast crises. The K-562 cell line
has previously been described as exhibiting a high concentration of cells
aneuploid in chromosome 8.
A second cell line, GMO6912A, was obtained from the NIGMS Human Genetic
Mutant Cell Repository in Camden, New Jersey, Depository Number GMO6912A.
The GMO6912A cell line was established from a seventeen year old male, is
not known to exhibit any significant aneuploidy in chromosome 8, and has
been characterized as exhibiting fragile X syndrome.
The K-562 cell line was used as a starting material to produce the first
cell source of the invention and the GMO6912A cell line was used as the
starting material for the second cell source. Each of these cell lines
were separately treated as follows: initially the cell count of each of
the cell sources was determined. A 10 milliliter culture of each of the
cell lines in standard growth media, MEM, minimum essential media from
Gibco/BRL, was treated with 0.2 milliliters of colcemid to arrest cell
growth. The mixtures were then allowed to sit for fifteen minutes. Each
treated cell source was then centrifuged for ten minutes and aspirated to
remove remaining media. The resulting cell pellets from each cell line
were then resuspended in a total of about twenty to 30 ml of MEM to total
volume of approximately thirty milliliters. Cell counts of the resulting
solutions were determined on a hemocytometer after dilution of the cell
mixtures with phosphate buffered saline ("PBS") and trypan blue.
Sufficient PBS and trypan blue was added to produce a count of
approximately 30 to 100 cells per quadrant of the hemacytometer. The cells
were then counted in four quadrants in each of the two chambers of the
hemacytometer and recorded. The actual number of cells determined was then
multiplied by the dilution factor, i.e. the amount of PBS and trypan blue
added.
The cell count for the K-562 was determined to be 6.4.times.10.sup.5 cells
per cc. The cell count for the GMO6912A was determined to be
1.17.times.10.sup.6 cells per cc. The GMO6912A cell suspension was then
diluted with MEM to a calculated cell count the same as that for the K-562
cell suspension:
______________________________________
9cc (1.17 .times. 10.sup.6 /cc) + 7.5cc MEM =
1.053 .times. 10.sup.7 cells/16.5cc =
6.4 .times. 10.sup.5 cells per cc
______________________________________
Both the first and second cell sources thus had equivalent cell
concentrations. The two cell sources were then mixed to produce three cell
suspensions: (1) having about 5% of the cells exhibiting the trisomy 8 of
the K-562 cell line, (2) 10% exhibiting trisomy 8; and (3) about 40%
exhibiting trisomy 8. The mixtures in Example 1-2, 1-3 and 1-4 were
produced by mixing by volume, respectively, 1 part of the K-562 suspension
with 1 part of the GMO6912A suspension, 1 part of K-562 with 9 parts of
GMO6912A and 1 part K-562 with 18 parts GMO6192A. Table 1 shows the
resulting cell mixtures and controls.
TABLE 1
______________________________________
EXAMPLE PERCENT PERCENT
NUMBER TRISOMY 8* GMO6912A*
______________________________________
1-1 100 0
1-2 40 95
1-3 10 90
1-4 5 50
1-5 0 100
______________________________________
*Based on total cells
After the cell counts were determined and the resulting cell mixtures
produced, the mixtures were then immediately harvested by addition of 0.1
ml of colcemid for each approximately 5 ml by volume of the cell mixture
or control and allowed to stand for one hour. The mixtures and controls
were then incubated for 1 hour at 37.degree. C. in 5% CO.sub.2 /H.sub.2 O
solution. The thus treated cell mixtures and controls were then
centrifuged for 10 minutes and any remaining supernatent was also removed.
The cell mixtures and controls were then resuspended by the addition of 10
ml of 0.075M KCL and were incubated at 37.degree. in a water bath for 20
minutes. The cell mixtures and controls were then centrifuged again for 10
minutes and the supernatent removed. Two ml of Carnoy's fixative (3/1 by
volume methanol/acidic acid) was slowly added and mixed. An additional 6
ml of Carnoy's fixative was added and mixed to produce the fixed cell
mixtures and controls. The fixed cell mixtures and fixed cell controls
were then allowed to stand for 5 minutes at room temperature and then
centrifuged for 10 minutes. The supernatent was removed and the cells were
resuspended by the addition of 8.5 ml of Carnoy's fixative to each. The
mixtures and controls were again centrifuged for 10 minutes, the
supernatent was removed and sufficient additional Carnoy's fixative was
added to resuspend the pellet so that the resulting solutions were
slightly cloudy.
Hybridization slides (two slides per Example) were then prepared from each
of the Examples 1-1 through 1-5 as follows:
Glass microscope slides were dipped in 70% by volume ethanol/H.sub.2 O and
wiped dry with a wipe. Each slide was dipped briefly into a 3:1 by volume
ethanol:acetic acid solution. One to two drops of fixed cell suspension
was then immediately dropped onto the microscope slide using a pasteur
pipette. The slide was placed in a humidified environment of approximately
50% humidity and approximately 70.degree.-80.degree. C. The slide remained
in the humidified environment until the cell and fixative suspension dries
on the slide. Slide was removed from the humidified environment and then
placed at room temperature/environment for approximately 24 hours before
use.
FISH was then performed on each of the slides to determine percent
aneuploid cell content as follows:
Slides were placed into a denaturing solution of pH 7.5 comprising 70%
formamide and 30% 20 X aqueous sodium chloride/sodium citrate solution
("SSC"), which is 2.99M NaCl and 0.3M Sodium citrate, and maintained at a
temperature of 74.degree. C. for 5 minutes. Excess fluid was drained from
the slide after removal from the bath. Simultaneously with the slide
denaturation, a hybridization mixture comprising 7 microliters of
SpectrumCEP Hybridization Buffer, lot no. 9411, obtained from Imagenetics,
Framingham, Mass., 1 microliter of SpectrumCEP 8 Orange Chromosome
Enumeration Probe, lot number 9431, obtained from Imagenetics, Framingham,
Mass., and 2 microliters of H.sub.2 O was mixed briefly in a
microcentrifuge. The hybridization mixture was then denatured in a
74.degree. C. water bath for 5 minutes. The hybridization mixture was then
cooled on an ice bath for 1-5 minutes. The hybridization mixture was then
microcentrifuged briefly for approximately 2 seconds and was pipetted 3-5
times each before addition to the slides.
Immediately after removing the slides from the denaturing bath, the slides
were placed into 70% ethanol/water wash for 1 minute, removed, touched to
the side of a coplin jar maintained at 74.degree. C., placed into an 85%
ethanol/water wash for 1 minute, touched to the side of the jar and then
placed into a 100% ethanol wash for 1-5 minutes. The slides were then
dried, and excess ethanol was drained by blotting the edge of the slide
until all visible moisture was gone. The denatured slides were then placed
on a 45.degree. C. to 50.degree. C. slide warmer. The slides were
maintained on the warmer for at least 3 minutes before application of the
hybridization mixture.
To each of the slides, 10 microliters of the hybridization mixture was
applied by pipetting directly onto the target. A cover slip was then
placed over the hybridization mixture, avoiding formation of bubbles under
the cover slip. Rubber cement solution was applied around all edges of the
cover slip. The slides were then incubated in an air incubator at
42.degree. C. overnight by placement in an air tight box containing a
piece of blotting paper wetted with 3-5 drops of water.
Wash solutions for the post hybridization treatment of the slides were all
maintained at 47.degree. C. The slides were removed from the incubator and
cover slips removed and placed in a first water bath containing a wash
solution of 50% 2 X SSC (0.3M NaGl and 0.03M sodium citrate) and 50%
formamide. The slides were agitated gently and allowed to stand in the
wash solution for 10 minutes. The slides were removed from the first wash
and drained but not dried. The slides were then placed in a second wash
solution of the same make-up as the first wash solution for 10 minutes.
Again the slides were drained without allowing then to dry and transferred
to a third wash solution of the same make-up for 10 minutes. The slides
were again drained without allowing to dry. The slides were then
transferred to a fourth wash solution comprising 2 X SSC and allowed to
stand for 10 minutes. The slides were again drained without allowing to
dry and transferred to a fifth wash solution which comprised 2 X SSC and
0.1% detergent NP-40. The slides were allowed to stand in the fifth wash
solution for 5 minutes. The slides were then removed, drained thoroughly
and allowed to air dry in dim light. Ten microliters of standard
counterstain solution comprising 2',6-diamino-phenylindole (DAPI) and
p-phenylene diamine and glycerol were then applied to the target area of
the slide, and cover slips were placed on the target areas. The slides
were then left at room temperature until the hybridization results
observed.
Hybridization results were counted by an individual who was blinded to,
i.e. not involved in, the experimental preparation, counting 500 cell
nuclei per slide using a fluorescence microscope having a dual bandpass
DAPI/Spectrum Orange filter supplied by Imagenetics. The count determined
the number of cells having two or more fluorescent signals per nucleus.
Cells exhibiting trisomy 8 each have three fluorescent signals. A total of
two slides for each of Examples 1-1 through 1-5 was counted.
Table 2 shows the average of two slides, percent diploid chromosome 8 and
percent trisomy 8 determined for each of Examples 1-1 through 1-5, based
on percent of total cells counted per slide:
TABLE 2
______________________________________
PERCENT CELLS WITH
PERCENT CELLS WITH
EXAMPLE TWO SIGNALS THREE SIGNALS
NUMBER Actual Expected Actual Expected
______________________________________
1-1 8.3 -- 86.8 --
1-2 54.4 50.8 40.9 43.9
1-3 83.6 84.7 10.2 9.7
1-4 89.0 88.9 5.6 5.6
1-5 93.2 -- 1.1 --
______________________________________
The actual values of 2 and 3 signals in the 100% K-562 and 100% GMO6912A
were determined according to signal counts of the hybridization with the
SpectrumCEP8 probe.
The hybridization count data showed that the expected count was achieved
with less than 5% difference between the observed and expected
percentages. The control compositions of the invention prepared by
fixation of the cells only after mixing of the two cell sources, each of
which comprised cells which were still capable of multiplication, resulted
in reproducible artificial concentrations of aneuploid cells.
COMPARATIVE EXAMPLE 1
Comparative Example 1 describes Applicants' early attempts to prepare the
compositions of the invention by mixing of two cell sources, each
comprising fixed cells.
A fixed pellet of bone marrow exhibiting trisomy in various chromosomes was
obtained from The Mayo Clinic, Rochester, Minn. The pellet was washed two
times with Carnoy's fixative and stored in a refrigerator in 10 ml
fixative. Supernatent containing cells was removed and the suspension was
transferred to a new eppendorfer tube. The suspension was diluted by
adding 0.1 ml additional Carnoy's fixative solution for each 0.1 ml of the
cell suspension to produce a 1:10 dilution. The fixed cell suspension was
diluted 1:1 with trypan blue to enable ease of counting with the
hemocytometer. The cell counts were believed inaccurate because the
suspension was evaporating too quickly resulting in cell motion and due to
excessive clumping of the cells. Table 3 illustrates the variance in the
cell counts obtained from counting the resulting suspension. It was
expected that each quadrant would contain about the same number of cells
in each chamber.
TABLE 3
______________________________________
CHAMBER 1
CHAMBER 2
______________________________________
QUADRANT 1 49 53
QUADRANT 2 44 33
QUADRANT 3 76 63
QUADRANT 4 99 0
______________________________________
The average of counts in Chamber 1 was 67 cells compared to 49.6 in Chamber
2. Thus another method to obtain a better count of the fixed bone marrow
trisomy sample was tried. Applicants then tried a 1:10 dilution of the
fixed cells with phosphate buffered saline solution ("PBS"). The resulting
suspensions were then attempted to be counted. The evaporation problem
seemed to alleviate by dissolving the fixed cells in PBS. However, the
cell clumping continued and accurate counts were again not obtained.
Because accurate cell counts are necessary for each of the two cell sources
to permit mixing to achieve the desired amount of cells containing a
specific abnormality. Applicants determined that it was simply not
feasible to start with two fixed cell sources before mixing due to the
cell clumping problem.
EXAMPLE 2
A separate set of control compositions using the K-562 and GMO6912A cell
sources were made to examine reproducibility of the manufacture. The same
procedure as used in Example 1 was followed.
A 1.0 ml of the K-562 cell suspension was diluted with 0.5 ml of trypan
blue and cell count was determined on a hemocytometer to be
4.1.times.10.sup.5 cells/cc. A 1.0 ml suspension of the GMO5912A cells was
diluted with 0.5 ml of trypan blue and the cell count was determined to be
3.93.times.10.sup.5 cells/cc. 9.0 ml of the K-562 suspension was then
diluted slightly with 0.4 ml of RPMI to achieve an equivalent cell count
to the GMO69 12A suspension.
The control compositions were then produced by mixing as follows:
Example 2-2:2 ml of K-562 suspension+1.5 ml GMO6912A suspension (about 50%
trisomy 8)
Example 2-3:0.5 ml of K-562+4.32 ml GMO6912A (10% trisomy 8)
Example 2-4:0.25 ml K-562+5.25 GMO6912A (5% trisomy 8)
Example 2-1 was the control using 100% K-562 suspension.
Example 2-5 was the control using 100% GMO6912A suspension.
All Examples were then harvested and suspended in Carnoy's fixative as in
Example 1 and hybridization slides were produced as in Example 1. Two
slides for each Example were made.
The slides were hybridized using the procedure of Example 1 with the same
hybridization mix of Example 1 using SpectrumCEP 8 Orange Chromosome
Enumeration Probe from Imagenetics.
The hybridization results were then counted, again using an observer
blinded to the hybridization procedure. Table 4 shows the hybridization
results.
TABLE 4
______________________________________
PERCENT CELLS WITH
PERCENT CELLS WITH
EXAMPLE TWO SIGNALS THREE SIGNALS
NUMBER Actual Expected Actual Expected
______________________________________
2-1 7.8 -- 86.1 --
2-2 47.57 -- 46.0 49.4
2-3 85.73 -- 9.1 11.1
2-4 88.8 -- 6.3 6.6
2-5 90.0 -- 4.5 --
______________________________________
To check reproducibility of the hybridization results, a second set of two
slides for each Example was then hybridized using the same procedure and
hybridization mix and the hybridization results determined. Table 5 lists
the results.
TABLE 5
______________________________________
PERCENT CELLS WITH
PERCENT CELLS WITH
EXAMPLE TWO SIGNALS THREE SIGNALS
NUMBER Actual Expected Actual Expected
______________________________________
2-1 8.13 -- 84.35 --
2-2 47.92 -- 44.9 49.4
2-3 83.36 -- 9.52 11.1
2-4 87.40 -- 6.2 6.6
2-5 93.4 -- 2.9 --
______________________________________
Comparing the results in Tables 4 and 5 shows good reproducibility for
hybridization of the control compositions. The inventive compositions are
reproducibly useful as control compositions for DNA chromosome enumeration
probes.
The above description should not be considering limiting for the scope of
the invention as other variations are possible. Rather, its scope is set
out in the following claims.
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